The allele-specific primer PCR (ASP-PCR) is a method used for the detection of mutations such as SNPs (single nucleotide polymorphisms). A typical allele-specific primer PCR uses a set of a primer that has a nucleotide corresponding to a mutated allele-specific nucleotide at the 3′ end thereof, and a primer that, in pairs with the primer, amplifies a region comprising an SNP site to amplify specifically a target polynucleotide comprising a mutated allele, thereby detecting the mutated allele (Japanese Patent No. 2853864).
In the allele-specific primer PCR method as described above, however, it is necessary to set strict PCR conditions for amplifying specifically the target polynucleotide comprising the mutated allele, and thus, there has been a problem that it is difficult to achieve a high detection sensitivity (JP-A-2010-35532).
As a technique for solving such a problem, there is known a nucleic acid amplification method, which is an application technique of the typical allele-specific primer PCR. The method is characterized by that PCR reaction for a target nucleic acid is performed using (i) a first forward primer having a nucleotide sequence identical to a partial region comprising an SNP site of the target nucleic acid, except for having a wild-type nucleotide as the SNP site at only one of the second nucleotide and the third nucleotide from the 3′ end, (ii) a second forward primer having a nucleotide sequence identical to the partial region comprising the SNP site of the target nucleic acid, except for having a mutated nucleotide as the SNP site at only one of the second nucleotide and the third nucleotide from the 3′ end, and (iii) a reverse primer having a nucleotide sequence identical to a partial region on the 5′ end side from the SNP site of a complementary chain of the target nucleic acid, the partial region not comprising a site corresponding to the SNP site (JP-A-2010-35532).